Compound for inhibiting binding between DX2 protein and p14/ARF protein, and pharmaceutical composition for treating or preventing cancer disease containing same as effective ingredient

ABSTRACT

Disclosed is a new compound that inhibits binding between a DX2 protein and a p14/ARF protein, a pharmaceutical composition including the new compound as an effective component for treating or preventing a cancer disease, an anticancer adjuvant for improving an anticancer effect of a drug-resistant anticancer drug, and a composition including an AIMP2-DX2 protein or a fragment thereof for diagnosing lung cancer.

CROSS REFERENCE TO PRIOR APPLICATION

This application is a divisional Application of U.S. patent applicationSer. No. 15/323,099 filed on Dec. 30, 2016 under 35 U.S.C. § 120, whichis the 35 U.S.C. § 371 national stage of International applicationPCT/KR2015/006701 filed on Jun. 30, 2015, which claims priority toKorean applications 10-2015-0092057 filed on Jun. 29, 2015 and10-2014-0081396 filed on Jun. 30, 2014, respectively.

BACKGROUND

The present invention relates to a new compound that inhibits bindingbetween a DX2 protein and a p14/ARF protein, a pharmaceuticalcomposition including the new compound as an effective component fortreating or preventing a cancer disease, an anticancer adjuvant forimproving an anticancer effect of a drug-resistant anticancer drug, anda composition including an AIMP2-DX2 protein or a fragment thereof fordiagnosing lung cancer.

Lung cancer is mainly caused by carcinogens, and incidence of lungcancer has been on a rising trend worldwide. In South Korea, lung cancerhaving the highest mortality rate among all cancers is regarded as oneof the most serious diseases. Lung cancer is divided into two groups,small cell lung cancer (SCLC) and non small cell lung cancer (NSCLC),and the NSCLC is also divided into sub-groups: adenocarcinoma, squamouscarcinoma, large cell carcinoma, and adenosquamous carcinoma, dependingon lung tissue types. Clinical manifestations, such as areas prone tooccur depending on different lung tissue types, progression type andspeed, and symptoms, may vary as well as treatment methods.

Most of lung cancers cannot be treated by chemotherapy and radiationtherapy. Chemotherapy and radiation therapy may be used by reducing asize of SCLC while complete treatment cannot be expected fromchemotherapy and radiation therapy. Since an anticancer drug is lesseffective in NSCLC than SCLC, the treatment of lung cancer usingchemotherapy only is almost impossible. Instead, complete removal oftumors in a surgical manner is the only effective treatment. However,about 30% or less of patients with lung cancer have tumors that cannotbe totally resected when being diagnosed. In addition, only one-third orless of the patients survive for 5 years after surgical resection isdone.

Therefore, there is a great demand for a method of more accuratelydetecting the early diagnosis of lung cancer and the spread of cancerand more effectively treating lung cancer.

SUMMARY

An object of the present invention is to provide a new compound thatinhibits binding between a DX2 protein and a p14/ARF protein inconsideration of development of a new useful compound for treatment of acancer disease including lung cancer.

In addition, another object of the present invention is to provide apharmaceutical composition for treating or preventing a cancer disease,the pharmaceutical composition including, as an effective compound, acompound that inhibits binding between a DX2 protein and a p14/ARFprotein.

In addition, another object of the present invention is to provide ananticancer adjuvant for improving an anticancer effect of adrug-resistant anticancer drug, the anticancer adjuvant including, as aneffective component, a compound that inhibits binding between a DX2protein and a p14/ARF protein.

In addition, another object of the present invention is to provide acomposition for diagnosing lung cancer, the composition including anAIMP2-DX2 protein or a fragment thereof.

To achieve the objectives above, the present invention provides acompound represented by Formula 1 or 2:

In addition, the present invention provides a pharmaceutical compositionfor treating or preventing a cancer disease, the pharmaceuticalcomposition including, as an effective component, a compound representedby Formula 1, 2, or 3:

In addition, the present invention provides a pharmaceutical compositionfor inhibiting drug resistance of an anticancer drug, the pharmaceuticalcomposition including, as an effective component, a compound representedby Formula 1, 2, or 3.

In addition, the present invention provides a pharmaceutical compositionfor treating or preventing a cancer disease, the pharmaceuticalcomposition including, as effective components, a compound representedby Formula 1, 2, or 3 and an anticancer drug.

In addition, the present invention provides a composition for diagnosinglung cancer, the composition including an AIMP2-DX2 protein or afragment thereof.

In addition, the present invention provides a method of detecting anautoantibody to an AIMP2-DX2 protein to provide information for lungcancer diagnosis, the method including: (a) detecting an antibody to anAIMP2-DX2 protein from a sample derived from a subject; and (b)determining that a subject has lung cancer or is susceptible to lungcancer if an amount of the detected antibody in the subject increases ascompared with a normal person.

In addition, the present invention provides a kit for diagnosing lungcancer, the kit including an AIMP2-DX2 protein or a fragment thereof.

According to the present invention, a compound that inhibits interactionof a DX2 protein with a p14/ARF protein may be selected. An analogouscompound to the selected compound is synthesized, and a review of invitro and in vivo anticancer effects of the analogous compound showsthat the compound has an excellent anticancer effect. In particular, ina cell line which is resistant to an anticancer drug such as Adriamycin,it is confirmed that the a compound that inhibits interaction between aDX2 protein and a p14/ARF protein is treated so that resistance of ananticancer drug against Adriamycin is inhibited while an anticancereffect of the anticancer drug is improved. Accordingly, the disclosedcompound that inhibits interaction between a DX2 protein and a p14/ARFprotein can be significantly used as an anticancer drug or anticanceradjuvant for a cancer disease such as lung cancer.

In addition, the present invention provides a composition and a kit,each of which includes an AIMP2-DX2 protein or a fragment thereof fordiagnosing lung cancer. The composition and the kit may be used toidentify incidence of lung cancer only by using a serum sample of asubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an ELISA-based screening method for screening a specificinhibitor of DX2 and p14/ARF binding;

FIG. 2 illustrates A showing results of GST pull-down assay, B showingresults of immunoprecipitation analysis, and C showing results obtainedby performing protein binding analysis using a His-DX2 protein as aninhibitory effect of SLCB050 on DX2-p14/ARF binding (IP: a group bindingto a protein for precipitation, Sup: a group not binding to a proteinfrom which supernatant of the precipitate is separated, Input: celldebris);

FIG. 3 illustrates A, B, C showing results of GST pull-down assay toconfirm interaction of SLCB050 with a DX2-specific region, and D, Eshowing results of GST pull-down assay to confirm inhibitory effects ofHJH141204, HJH141206, and SLCB36 on DX2-p14/ARF binding (PPT: a groupbinding to a protein for precipitation, Sup: a group not binding to aprotein from which supernatant of the precipitate is separated);

FIG. 4 illustrates A showing dissociation of DX2 and p14/ARF afterperforming SLCB050 treatment, B showing differences in localization ofDX2 and p14/ARF after SLCB050 treatment, C showing SLCB050 treatmenteffects on NSCLC cell line H1299 and SCLC cell line H69, and D showingSLCB050 treatment effects on p14/ARF-deficient H322 cells;

FIG. 5 shows viability of various human lung cancer cell lines obtainedafter performing SLCB050 treatment thereon.

FIG. 6 shows results of soft agar colony formation assay obtained afterH128 cells which are SCLC cell lines were treated with SLCB050 treatmenton;

FIG. 7 shows viability of H128 cells which are SCLC cell lines after thecells were treated with SLCB050, HJH141204, HJH141206, and SLCB36;

FIG. 8 shows effects of SLCB050 treatment on reduction of drugresistance of an anticancer drug;

FIG. 9 shows inhibitory effects on drug resistance to Adriamycin in axenograft model using H446 (Adr: 0.2 μg/ml, SLCB050: 10 mg/kg);

FIG. 10 shows in vivo anticancer effects of SLCB050;

FIG. 11 shows hematoxylin-eosin (H-E) staining results of lung tissuesderived from SLCB050-treated DK mice;

FIG. 12 shows in vivo anticancer effects of SLCB050;

FIG. 13 shows the mechanism of SLCB050 of the present invention, whichis a compound inhibiting DX2-p14/ARF binding, on therapeutic effect onlung cancer;

FIG. 14 shows results of measuring AIMP2-DX2 expression levels invarious lung cancer cell lines (NSCLC: non small cell lung cancer cellline; SCLC: small cell lung cancer cell line; H322 and H460: NSCLC celllines; H146 and H69: SCLC cell lines; AIMP2: aminoacyl tRNA synthetasecomplex-interacting multifunctional protein 2; and Actin: Actin);

FIG. 15 shows results of measuring AIMP2-DX2 expression levels invarious lung cancer cell lines (NSCLC: non small cell lung cancer cellline; SCLC: small cell lung cancer cell line; A549, H1299, H32, H322,and H460: NSCLC cell lines; H146 and H69: SCLC cell lines; AIMP2:aminoacyl tRNA synthetase complex-interacting multifunctional protein 2;GAPDH: Glyceraldehyde 3-phosphate dehydrogenase);

FIG. 16 is a schematic diagram of an experimental process for measuringATMP2-DX2 autoantibody levels in serum of a patient with SCLC and NSCLS,wherein the experimental process consists of steps of preparing adiagnostic membrane (step 1), allowing a reaction with serum of apatient (stpep2), and allowing a reaction with anti-human antibody; and

FIG. 17 shows results of measuring AIMP2-DX2 autoantibody levels inserum of a patient with SCLC and NSCLS (Normal serum, N1˜N10: serum of anormal person; SCLC serum, S1˜S10: serum of a patient with SCLC; NSCLC,L1˜L20: serum of a patient with NSCLC; Lamin A: lamin A; and red colorrefers to a sample from which anti-AIMP2-DX2 antibodies were detectedwhile black color refers to a sample from which anti-AIMP2-DX2antibodies were not detected).

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in detail. KoreanPatent Application No. 2014-0113543 discloses that DX2 inhibitsinduction of carcinogen-inducible p14/ARF so that a specific bindinginhibitor of DX2 and p14/ARF can be used as an anticancer drug. In thisregard, the present invention is completed by discovering a specificbinding inhibitor of DX2 and p14/ARF and confirming an anticancer effectof the inhibitor.

The present invention provides a compound represented by Formula 1 or 2:

Here, the compound represented by Formula 1 is named as HJH141204, andthe compound represented by Formula 2 is named as HJH141206. Thesecompounds may inhibit binding between a DX2 protein and a p14/ARF.

In addition, a compound represented by Formula 3 is named as SLCB050.

In detail, referring to drawings, the compound according to anembodiment of the present invention which is a specific bindinginhibitor of DX2 and p14/ARF was screened by using an ELISA-basedscreening method of FIG. 1. Based on the screening reaction system,increases in ELISA values with increasing concentrations of p14/ARF weremeasured. However, in the case the treatment of a specific bindinginhibitor of DX2 and p14/ARF, such as SLCB050, ELISA reaction wasconsiderably reduced. Thus, by using the ELISA-based screening method,compounds that are specific binding inhibitors of DX2 and p14/ARF wereprimarily screened.

FIG. 2A confirms that SLCB050 completely blocked the DX2-p14/ARF bindingthrough GST pull-down assay. FIG. 2B confirms that SLCB050 inhibited theinteraction of p14/ARF with DX2 in cells through immunoprecipitationanalysis. Here, contransfected 293 cells were allowed to react withMG132 before immunoprecipitation analysis to prevent reduction of twoproteins, and after performing SLCB050 treatment (10 μM, 6 hours),immunoprecipitation assay was performed on the cells by using Mycantibodies (DX2). FIG. 2C shows that binding between DX2 and AIMP2 wasreduced by SLCB050 treatment in protein binding assay using His-DX2protein, in terms of inhibitory effect of SLCB050 on the binding betweenDX2 and AIMP2. In addition, since SLCB050 selectively inhibitedDX2-p14/ARF binding as shown in FIG. 3A, but not on p53-AIMP2 or DX2binding as shown in FIG. 3B and p14/ARF binding as shown in FIG. 3C, itwas confirmed that SLCB050 would be interacted with DX2-specific region.

In addition, HJH141204, HJH141206, and SLCB36, which are compoundssimilar to SLCB050, were each synthesized, and the GST pull-down assaywas performed thereon to verify inhibitory effect on DX2-p14/ARFbinding. As a result, as shown in FIGS. 3D and 3E, SLCB050, HJH141204,and HJH141206 showed inhibitory effects on DX2-p14/ARF binding and didnot affect the interaction of p53-p14/ARF, whereas SLCB36 did not showinhibitory effect on both binding. Accordingly, it was confirmed thatinclusion of a ribose ring structure in the compounds is required forthe binding inhibition.

FIG. 4A shows results obtained by detection of dissociation of DX2 andp14/ARF through immunoprecipitation analysis. Here, 293 cells weretransfected with indicating vectors for 24 hours and was allowed toreact with MG132 and SLCB050 for 6 hours. FIG. 4B shows differences inlocalization of DX2 and p14/ARF after the proteins were treated withSLCB050. Here, the interaction of DX2 with p14/ARF was blocked bySLCB050 treatment (10μM, 6 hours) so that DX2 was decreased and p14/ARFwas increased in the nucleoplasm. In addition, FIG. 4C shows SLCB050treatment effects on NSCLC cell line H1299 and SCLC cell line H69, andmore particularly, shows that SLCB050 treatment decreases DX2, butincreases p14/ARF in NSCLC cell line H1299 and SCLC cell line H69. Inaddition, FIG. 4D shows SLCB050 treatment effects on p14/ARF-deficientH322 cells. Here, DX2 levels decreased considerably in thep14/ARF-deficient H322 cells in response to SLCB050 treatment.

FIG. 5 shows viability of various human lung cancer cell lines after thecell lines were treated with SLCB050 for 24 hours, wherein the viabilitywas determined by MTT assay. Here, SCLC cells were very sensitive toSLCB050.

FIG. 6 shows results of soft agar colony formation assay. H128 cellswhich are SCLC cell lines were seeded in soft agar plates and allowed toreact with SLCB050 for 48 hours, and then, the cells were examined afterstaining the cells with trypan blue. SLCB050 reduced the number ofcolonies in a dose-dependent manner.

FIG. 7 shows viability of H128 cells which are SCLC cell lines after thecells were treated with SLCB050, HJH141204, HJH141206, and SLCB36. TheSLCB050, HJH141204, and HJH141206 treatments reduced the viability ofH128 cells in a dose-dependent manner, wherein the viability wasdetermined by MTT assay. Meanwhile, the SLCB36 treatment did notsignificantly affect the cell viability.

FIG. 8 shows inhibitory effect of SLCB050 on drug resistance of ananticancer drug against DX2 expressed cells. FIG. 8A shows effects ofSLCB050 on reduction of drug resistance of an anticancer drug whentreated together with GN25 [5 μM, 3-(5,8-dimethoxy-1,4-dioxonaphthalene-2-ylthio)propanoic acid] and SLCB050 (10 or 20 μM), which are p53activators, for 6 hours in DX2 and DK MEFs, wherein the effects weredetermined by MTT analysis. Here, resistance to GN25 in DX2 and DK cellswas abolished by SLCB050 treatment, thereby recovering anticancereffects of GN25. FIG. 8B shows effects of SLCB050 on reduction of drugresistance of primary tumor cells from DX2 and DK mice when treatedtogether with Adriamycin (0.2 μg/ml) and SLCB050 (10 μM) for 24 hours,wherein the effects were determined by MTT analysis. Here, resistance toAdriamycin was also abolished by SLCB050 treatment, thereby recoveringanticancer effects of Adriamycin. FIG. 8C shows sensitivity of SCLC celllines to combinational treatment with Adriamycin and SLCB050. Here, SCLCcell line H69 was partially responded to SLCB050 so that the sensitivityto Adriamycin improved, whereas p14/ARF-deficient H322 cells did notshow improved sensitivity by combinational treatment with SLCB050.

FIG. 9 shows inhibitory effects on drug resistance to Adriamycin in axenograft model using H446. FIG. 9A shows resistance to Adriamycin inthe xenograft model using H446, and also shows decreased resistance bySLCB050 treatment. FIG. 9B shows that tumor growth was suppressed within4 weeks by treatment of 5 mg/kg of Adriamycin, but the experiment wasceased by high toxicity. In constrast, low dose of Adriamycin (2.5mg/kg) with 10 mg/kg of SLCB050 obviously suppressed tumor growthwithout severe toxicity. FIG. 9C confirmed through Wester blotting thatcombinational treatment with Adriamycin and SLCB050 induced p53synergistically in primary tumor cells.

FIG. 10 shows in vivo anticancer effects of SLCB050. FIG. 10A shows adiagram of experimental schedule with DK mice in terms of examination ofin vivo anticancer effects. FIG. 10B shows hematoxylin-eosin (H-E)staining results of lung tissues derived from SLCB050-treated DK mice.Here, combinational treatment with Adriamycin and SLCB050 induced tumorregression. FIG. 10C shows that combinational treatment with Adriamycinand SLCB050 reduced tumor volumes.

FIG. 11 shows H-E staining results of lung tissues derived fromSLCB050-treated DK mice. Here, SCLC regions were more obviously erasedby combinational treatment with Adriamycin and SLCB050.

FIG. 12 shows in vivo anticancer effects of SLCB050. FIG. 12A is awestern blot analysis result showing reduction of DX2 in response toSLCB050 treatment. FIGS. 12B and 12C show TUNEL staining results fromlung tissues of DK mice treated with Adriamycin and SLCB050 incombination. Here, SLCB050 treatment obviously increased apoptosis tumercells.

As shown in FIG. 13, the results above indicate that DX2 produced byaberrant splicing of AIMP2 promotes tumor progression, in particularly,small cell lung cancer, via direct interaction and inhibition ofp14/ARF. In this regard, it was confirmed that such small cell lungcancer can be treated by using compounds that inhibit DX2-p14/ARFbinding.

Accordingly, the present invention provides a pharmaceutical compositionfor treating or preventing a cancer disease, the pharmaceuticalcomposition including, as an effective component, a compound representedby Formula 1, 2, or 3:

The compound above may inhibit binding between a DX2 protein and ap14/ARF protein.

The cancer disease may be selected from the group consisting of lungcancer, colorectal cancer, liver cancer, stomach cancer, esophagealcancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladdercancer, prostate cancer, testis cancer, uterine cervical cancer,endometrial cancer, choriocarcinoma, ovarian cancer, breast cancer,thyroid cancer, brain cancer, head and neck cancer, malignant melanoma,lymphoma, and hematologic malignancy, and the lung cancer may be nonsmall cell lung cancer or small cell lung cancer, and more preferable,may be small cell lung cancer.

In addition, the present invention provides a pharmaceutical compositionfor inhibiting drug resistance of an anticancer drug, the pharmaceuticalcomposition including, as an effective component, a compound representedby Formula 1, 2, or 3.

The anticancer drug may be selected from the group consisting ofAdriamycin, Capecitabine, Caboplatin, Cisplatin, Oxaliplain,Cyclophosphamide, Docetaxel, Paclitaxel, Doxorubicin, Daunorubicin,Epirubicin, Idarubicin, Valrubicin, Mitoxantrone, Curcumin, Gefitinib,Erlotinib, Irinotecan, Topotecan, Vinblastine, Vincristine, Gemsitabin,Methotrexate, Trastzumab, Vinorelbine, Fluorouracil, and 3-(5,8-dimethoxy-1,4-dioxonaphthalene-2-ylthio)propanoic acid.

The compound above may inhibit binding between a DX2 protein and ap14/ARF protein to thereby inhibit drug resistance of the anticancerdrug and improve anticancer effect of the anticancer drug.

In addition, the present invention provides a pharmaceutical compositionfor treating or preventing a cancer disease, the pharmaceuticalcomposition including, as effective components, a compound representedby Formula 1, 2, or 3 and an anticancer drug.

The anticancer drug may be selected from the group consisting ofAdriamycin, Capecitabine, Caboplatin, Cisplatin, Oxaliplain,Cyclophosphamide, Docetaxel, Paclitaxel, Doxorubicin, Daunorubicin,Epirubicin, Idarubicin, Valrubicin, Mitoxantrone, Curcumin, Gefitinib,Erlotinib, Irinotecan, Topotecan, Vinblastine, Vincristine, Gemsitabin,Methotrexate, Trastzumab, Vinorelbine, Fluorouracil, and3-(5,8-dimethoxy-1,4-dioxonaphthalene-2-ylthio)propanoic acid, lungcancer, colorectal cancer, liver cancer, stomach cancer, esophagealcancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladdercancer, prostate cancer, testis cancer, uterine cervical cancer,endometrial cancer, choriocarcinoma, ovarian cancer, breast cancer,thyroid cancer, brain cancer, head and neck cancer, malignant melanoma,lymphoma, and hematologic malignancy, and the lung cancer may be nonsmall cell lung cancer or small cell lung cancer, and more preferable,may be small cell lung cancer.

The pharmaceutical composition may include, as a specific inhibitoragainst binding between DX2 and p14/ARF, not only screened compounds,SLCB050, HJH141204, and HJH141206, but also a pharmaceuticallyacceptable carrier of the screened compounds, in an effective amount.The term “effective amount” as used herein refers to an amountsufficient to exhibit therapeutic effects on cancer.

The pharmaceutically acceptable carrier included in the pharmaceuticalcomposition of the present invention may be any material conventionallyused at the time of formulation, and examples thereof include acarbohydrate compound (e.g., lactose, amylase, dextrose, sucrose,sorbitol, mannitol, startch, cellulose, etc), Acacia rubber, calciumphosphate, alginate, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution,alcohol, gum Arabic, vegetable oil (e.g., corn oil, cotton seed oil,soybean oil, olive oil, coconut oil, etc), polyethylene glycol, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesiumstearate, and mineral oil. But the examples of the pharmaceuticallyacceptable carrier re not limited thereto.

The pharmaceutical composition according to the present invention mayfurther include, in addition to the components described above, alubricant, a wetting agent, a sweetener, a flavoring agent, anemulsifying agent, a suspending agent, or a preservative. Materialssuitable as the pharmaceutically acceptable carrier and formulation areprovided in detail in Remington's Pharmaceutical Sciences (19th ed.,1995).

The pharmaceutical composition according to the present invention may beadministered orally or parenterally. In the case of parenteraladministration, the administration may be done via intravenousinjection, subcutaneous injection, or muscle injection.

A suitable dose of the pharmaceutical composition according to thepresent invention may vary depending on various factors such as a methodfor formulation, a method for administration, a patient's age, weight,sex, disease condition, or diet, administration time, an administrationroute, an excretion rate, and responsiveness. In general, a skilleddoctor may readily determine and prescribe an effective dose in terms ofdesired treatment or prophylaxis. According to a preferable embodimentof the present invention, the dose of the pharmaceutical composition maybe 0.0001 to about 100 mg/kg (body weight) per day, and it may beadministered once or several times per day.

The pharmaceutical composition according to the present invention may beprepared according to a method that can be easily carried out by thoseskilled in the art. For example, the pharmaceutical composition may beprepared in unit dosage form by formulation with a pharmaceuticallyacceptable carrier and/or excipient. Alternatively, the pharmaceuticalcomposition may be prepared in a multi-dose container. Here, aformulation of the pharmaceutical composition may be oil or solution inan aqueous medium, suspension or emulsion, extract, powder, granule,tablet, or capsule, and may further include a dispersant or astabilizer.

The present invention provides a composition for diagnosing lung cancer,the composition including AIMP2-DX2 protein or a fragment thereof.

Autoantibodies to AIMP2-DX2 are produced in a patient with lung cancer,especially with small cell lung cancer. In this regard, detection ofautoantibodies enables diagnose of lung cancer. As a representativemethod of detecting the autoantibodies, target proteins to be antigensare immobilized on a fixture to be then reactive with a blood or serumsample including antibodies extracted from a subject, thereby confirmingthe presence of binding between the autoantibodies and the sample.

The inventor of the present invention discovered the production ofautoantibodies to AIMP2-DX2 in a patient with lung cancer, and confirmedwhether autoantibodies to AIMP2-DX2 are found in serum of a subjectafter immobilizing AIMP2-DX2 on a fixture for reaction with serum of thesubject. Here, autoantibodies to AIMP2-DX2 were not detected in serum ofa healthy person while the autoantibodies were detected in a patientwith SCLC or NSCLC.

Therefore, AIMP2-DX2 or a fragment thereof may be used for diagnosis oflung cancer. AIMP2-DX2 may be a deletion variant of AIMP2 lacking exon 2from the AIMP2 sequence, and ARS-interacting multi-functional protein 2(AIMP2) is one of proteins related to formation of an aminoacyl-tRNAsynthetase (ARS) composite, and is also referred to as p38/JTV-1 or p38.

The inventor of the present invention discovered in a previous studythat, in terms of a new function of AIMP2, genetic collapse of AIMP2induced overexpression of c-myc and accordingly causedhyperproliferation of alveolar epithelial cells in lungs, therebyinducing neonatal lethality of neuronal mice. In addition, the inventorof the present invention discovered that AIMP2 induced by TGF-β moves tothe nucleus for inhibition of expression based on molecular andcytological analysis (M. J. Kim, B.-J. Park, Y.-S. Kang, H. J. Kim,J.-H. Park, J. W. Kang, S. W. Lee, J. M. Han, H.-W. Lee, S. Kim, Nat.Genet. 34, 330-336, 2003).

AIMP2-DX2 of the present invention may be a deletion variant of AIMP2lacking exon 2 from the AIMP2 sequence. The AIMP2 sequence is found inseveral databases (312aa version:AAC50391.1 or GI:1215669; 320aaversion: AAH13630.1, GI:15489023, BC013630.1) and publications (312aaversion: Nicolaides, N. C., Kinzler, K. W. and Vogelstein, B. Analysisof the 5′ region of PMS2 reveals heterogeneous transcripts and a noveloverlapping gene, Genomics 29 (2), 329-334 (1995)//320 aa version:Generation and initial analysis of more than 15,000 full-length humanand mouse cDNA sequences, Proc. Natl. Acad. Sci. U.S.A. 99 (26),16899-16903 (2002)). AIMP2-DX2 is a protein lacking a regioncorresponding to exon 2 in the sequence. KR 2004-0078035 disclosed bythe same inventor of the present invention discloses cancer therapyefficacy of AIMP2, and the description of AIMP2 in this patent documentis incorporated herein by reference.

AIMP2-DX2 protein may include a protein lacking exon 2 from the wholeAIMP2 sequence, and in this regard, may also include a protein lackingexon 2 from AIMP2 equivalents (functional equivalents that are variantsresulting from substitution, deletion, insertion, or a combinationthereof of the AIMP2 amino acid sequence, but have substantiallyequivalent activity to AIMP2, or functional derivatives that havemodifications that enhance or reduce physicochemical properties orhaving substantially equivalent activity to AIMP2) is deleted.

AIMP2-DX2 protein may include a protein in which the amino acid sequencespanning exon 2 in AIMP2 wholly deleted, a protein including the aminoacid sequences of exon 2 so that only a part of the amino acid sequencespanning exon 2 is deleted from exon 1, exon 3, exon 4, or all of theseexons, a protein in which the amino acid sequence of exon 2 in AIMP2 ispartially deleted. Preferably, AIMP2-DX2 of the present invention mayinclude a protein in which the amino acid sequence spanning exon 2 inAIMP2 is wholly deleted.

AIMP2-DX2 protein of the present invention may include not only aprotein having natural-occurring amino acid sequences, but also avariant of modified sequences. The variant of AIMP2-DX2refers to aprotein having a different sequence from a natural-occurring amino acidsequence of AIMP2-DX2 prepared by deletion, insertion, non-conserved orconserved substitution, or a combination thereof. The alteration ofamino acids in proteins and peptides where molecular activity is notsubstantially impaired is well known in the art. The most commonalternation includes alternation between amino acid residues Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

In some cases, AIMP2-DX2 protein may be subjected to modifications suchas phosphorylation, sulfation, acrylation, glycosylation, methylation,and farnesylation.

The fragment of AIMP2-DX2 protein of the present invention refers to apeptide or protein including a part of the amino acid sequences ofAIMP2-DX2 protein. Then, any material having a part of AIMP2-DX2 aminoacid sequence and a structure allowing binding of an antibodyspecifically to AIMP2-DX2 protein may be used as the fragment ofAIMP2-DX2 protein.

AIMP2-DX2 or a fragment thereof may be naturally extracted, synthesized,or prepared by recombinant methods based on DNA sequences. When arecombinant DNA technology is used, a suitable expression vectorcarrying a nucleic acid encoding AIMP2-DX2 was transformed to arecombinant expression vector, and a host cell was cultured with thetransformant to allow expression of AIMP2-DX2, thereby coveringAIMP2-DX2 from the transformant.

Preferably, AIMP2-DX2of the present invention may include an amino acidsequence of SEQ ID NO: 5 or 6.

The lung cancer of the present invention may include both non small celllung cancer (NSCLC) and (small cell lung cancer (SCLC), and preferably,the lung cancer of the present invention may include SCLC.

The present invention provides a method of detecting anautoantibody toAIMP2-DX2 protein to provide information for lung cancer diagnosis, themethod including: (a) detecting an antibody to AIMP2-DX2in a samplederived from a subject; and (b) determining that a subject has lungcancer or is susceptible to lung cancer in the case when an amount ofthe detected antibody in the sample is greater than that in a normalperson.

Hereinafter, the method of the present invention will be described stepby step.

(a) Detecting an Antibody to AIMP2-DX2from a Sample Derived From aSubject

In step (a), an antibody to AIMP2-DX2 protein is detected from a samplederived from a subject.

The sample derived from the subject refers to a substance extracted froma person desire to confirm whether the person has lung cancer, and typesof the substance are not particularly limited.

The sample of the present invention refers to, for example, acomposition obtained or derived from a subject desire to confirm whetherthe person has lung cancer, the composition including autoantibodiesthat can be identified based on physical, biochemical, or physiologicalcharacteristics.

The sample may include blood of biological origin and other liquid andtissue samples. The source of the tissue sample may include fresh,frozen, and/or preserved organs or tissue samples, solid tissues frombiopsy or aspiration, blood, any blood component, body fluid, and cellsor serum from any time during pregnancy or embryogeny of a subject. Thesample of the present invention may include whole blood, blood-derivedcells, serum, plasma, lymph, synovia, cell extract, and a combinationthereof, but is not limited thereto. Preferably, the sample may includeserum, plasma or serum, or more preferably, serum.

The antibody may be against AIMP2-DX2 protein, and more preferably, maybe an anti-AIMP2-DX2 autoantibody, and more preferably, may be ananti-AIMP2-DX2 IgG.

The detecting of the antibody to AIMP2-DX2 may be preferably made byantigen-antibody binding. That is, an antibody specifically binds toepitopes which are two-dimensional to three-dimensional parts of anantigen. Therefore, if an antigen and an antibody are able to bind toeach other immunologically, it is said that the antigen and the antibodyare “specific”, “recognizable”, or “bound”. The antigen may bepreferably AIMP2-DX2 protein or a fragment thereof.

The detecting of the antibody of the present invention may be performedby any known method of detecting the presence of antibody. However, sucha known method may be heterogeneous or homogeneous, sequential orsimultaneous, or competitive or non-competitive. The detecting of theantibody of the present invention may be performed in a quantitativemanner to test a concentration or amount of anti-AIMP2-DX2 autoantibody.Alternatively, the detecting of the antibody of the present inventionmay be performed in a qualitative manner to test the presence ofanti-AIMP2-DX2 autoantibody.

Preferably, the step (a) of detecting the antibody to AIMP2-DX2from thesample derived from the subject may include: (a1) applying the samplederived from the sample to a fixture on which AIMP2-DX2or a fragmentthereof is immobilized to allow a reaction; and (a2) detectingantibodies bound to AIMP2-DX2in the reaction product.

The detecting of the antibody of the present invention may be performedby both binding between a non-immobilized antigen and an antibody andbinding between an immobilized antigen and an antibody, but morepreferably, may be performed by binding between a non-immobilizedantigen and an antibody.

That is, after AIMP2-DX2(antigen) that is specifically reactive to ananti-AIMP2-DX2 autoantibody is bound to a fixture (solid support), thesample derived from the subject is mixed the reaction product to testthe presence of the anti-AIMP2-DX2 antibody. Here, the antigen which isin a bound state with the fixture is in contact with a biological samplewhich is an unbound state, thereby forming antigen-antibody binding.Afterwards, the fixture is washed so that the reaction sample includingantibodies that are not bound to immobilized antigens is removed. Aftercompletion of such treatments, an immune complex of the antigen and theanti-AIMP2-DX2 antibody is formed.

The detection of the present invention may be performed by, morepreferably, a sandwich assay including procedures of adding a secondaryantibody conjugate labeled with a detectable marker (for example,anti-human IgG antibody) to form a sandwich-type antigen-antibodycomplex through induction of binding between the secondary antibodyconjugate and the immune complex and identifying the detectable markerconjugated to the secondary antibody bound to the sandwich-typeantigen-antibody complex.

The secondary antibody may include an immunoglobulin fragment preparedby recombination with natural immunoglobulines isolated from non-humanprimates (e.g., anti-human IgG mouse antibody, anti-human IgG goatantibody, etc) or by synthesis.

The marker conjugated to the secondary antibody may be, preferably, aconventional coloring agent that performs a color reaction, and examplesof the marker include a fluorescein, such as horseradish peroxidase(HRP), alkaline phosphatase, coloid gold, poly L-lysine-fluoresceinisothiocyanate (FITC), and Rhodamine-B-isothiocyanate (RITC), and a dye.

The sandwich assay was disclosed in U.S. Pat. No. 5,876,935, andidentifying of the detectable marker conjugated to the secondaryantibody may be performed by conventional methods such as enzyme-linkedimmunosorbent assay (ELISA), radioimmnoassay (RIA), sandwich assay,western blotting on polyacrylamide gel, immunoblotting analysis, orimmnohistochemical staining.

As the fixture of the present invention, any solid support widely usedas a means for immobilizing AIMP2-DX2or its fragment may be used.Examples of suitable materials for use as the fixture include syntheticmaterials, such as polystyrene, polyvinylchloride, and polyamide, andother synthetic polymers. In addition, such materials may includenatural polymers, such as cellulose, or may be derived from naturalpolymers, such as cellulose acetate, nitrocellulose, and glass. Thefixture may be in form of ball, branch, tube, and microassay ormicrotiter plate. In addition, such materials may have a sheet-typestructure, such as a paper strip, a small plate. And a membrane.

(b) Determining That a Subject Has Lung Cancer or is Susceptible to LungCancer in the Case When an Amount of the Detected Antibody in the Sampleis Greater Than That in a Normal Person

In step (b), a subject with an increased amount of antibodies detectedin step (a) as compared with an amount of anti-AIMP2-DX2 antibodies in anormal person is determined to have lung caner or be susceptible to lungcancer.

The amount of antibodies detected in step (a) refers to an amount ofantibodies to AIMP2-DX2 protein in the sample, i.e., an amount ofantu-AIMP2-DX2 autoantibodies.

The anti-AIMP2-DX2 autoantibodies are not detected in a normal personwho does not have lung cancer, but are detected in a patient with lungcancer. In particular, the anti-AIMP2-DX2 autoantibodies aresignificantly highly detected in a patient with SCLC.

Therefore, when the level of autoantibodies to AIMP2-DX2 proteindetected in a sample of a subject is higher that of autoantibodiesdetected in a sample of a normal person, the target subject may bedetermined to have lung cancer or to be highly susceptible to lungcancer.

Referring to the steps described above, the method of detectingautoantibodies to AIMP2-DX2 protein of the present invention may provideinformation for lung cancer diagnosis to the target subject.

According to an embodiment of the present invention, various SCLC andNSCLC cell lines were incubated, and transcription and expression levelsof AIMP2-DX2 were measured. As a result, the transcription andexpression of AIMP2-DX2 in SCLC and NSCLC cell lines were confirmedwithout a difference in levels.

According to an embodiment of the present invention, serum of a patientwith SCLC and NSCLC was ensured to measure an amount of anti-AIMP2-DX2antibodies in the serum by using a nitrocellulose membrane to whichAIMP2-DX2 protein is attached. Consequently, it was confirmed that theanti-AIMP2-DX2 antibodies were not detected in a control group includingserum of a healthy person, whereas the anti-AIMP2-DX2 antibodies weredetected in a patient with SCLC and NSCLC. In particular, in the case ofa patient with SCLC, it was confirmed that the anti-AIMP2-DX2 antibodiesare significantly highly detected.

Meanwhile, the present invention provides a kit for diagnosing lungcancer, the kit including AIMP2-DX2 protein its a fragment.

The kit of the present invention may include AIMP2-DX2 protein or itsfragment, detect antibodies to AIMP2-DX2 protein in the sample, andenable to diagnose lung cancer of a subject.

The kit of the present invention is characterized by including AIMP2-DX2protein or a fragment thereof, and preferably, may be provided in afixed conditionon a fixture (solid support) on which AIMP2-DX2or itsfragment can be fixed. The fixture may be the same as defined above.

In addition, the kit of the present invention may specifically bind toan autoantibody binding to AIMP2-DX2, and may further include asecondary antibody conjugate to which a detectable marker is conjugated.The secondary antibody may be an antibody binding to an autoantibody(anti-AIMP2-DX2 antibody), and preferably, may be an anti-human IgGantibody. The detectable marker may be the same as defined above.

In addition, the kit of the present invention may further include ananti-AIMP2-DX2 autoantibody with a known amount in advance. Suchanti-AIMP2-DX2 autoantibody with a known amount in advance may be usedto establish a standard curve for measurement of an amount of ananti-AIMP2-DX2 autoantibody with an unknown amount in a sample derivedfrom a subject.

In addition, the kit of the present invention may include a suitablesubstrate in terms of an antigen-antibody reaction and color developmentof the marker, a buffer, or the like.

Hereinafter, the present invention will be described in detail byexplaining preferred embodiments of the invention. However, thepreferred embodiments should be considered in descriptive sense only andnot for purposes of limitation. Embodiments of the invention areprovided to more fully describe the present invention to one of ordinaryskill in the art.

EXAMPLE 1 Screening of an Inhibitor of DX2-p14/ARF Binding

The present invention is on the basis of KR 2014-0113543 disclosed bythe inventor of the present invention. Considering that DX2 inhibitsinduction of carcinogen-inducible p14/ARF so that a specific bindinginhibitor of DX2 and p14/ARF can be used as an anticancer drug, ananticancer drug was screened as follows.

1. Preparation of Compound Library

Compounds that were individually synthesized and natural compoundlibrary were prepared according to the related document (J. Clin. Oncol.16, 1207-1217, 1998; Nat. Rev. Cancer 2, 489-501, 2002), and 8,000compounds were provided from Korea Chemical Bank for the presentexperiments.

2. ELISA Analysis

To screen DX2-p14/ARF binding inhibitors, the screening system based onELISA modified as shown in FIG. 1 was used. That is, 0.5% His-DX2recombinant proteins were immobilized on a 96-well plate withparaformaldehyde (PFA). After performing drying and cleaning processes,GST-p14/ARF proteins were allowed to react with a random compound (finalconcentration: 0.1 mM). After 1 hour, the plate was washed using TBS-T,and then, incubated with anti-GST antibodies (1:10,000, 30 minutes) andanti-mouse-IgG-HRP (1:50,000, 1 hour). After performing a washingprocess twice, the plate was incubated with3,3′,5,5′-tetramethylbenzidine (TMB) solution (Calbiochem) and stopsolution (1N H₂SO₄) for a reaction. Afterwards, by using an ELISAreader, values were measured at 450 nm, thereby selecting candidate dugsas DX2-p14/ARF binding inhibitors. Here, more detailed protocol for thepresent experiment is described in the known related document (Nat. Rev.Cancer 2, 489-501, 2002), and PAK1-Smad4 binding inhibitors were used toexclude drugs that are confirmed as common inhibitors.

3. Recombinant Proteins, Immunoprecipitation, and GST Pull-Down Assays

Through GST pull-down assays, more specific binding inhibitors wereselected among the previously screened candidate drugs as DX2-p14/ARFbinding inhibitors. That is, a p14/ARF fragment (full-length) wasligated into EcoRI and Hind II sites of pGEX-TEV vector, which is amodified vector by adding a TEV protease cleavage site to pGEX-4T1. Therecombinant proteins were then expressed in E. coli strain BL21(DE3) asGST-fusion proteins. The proteins were purified by glutathione affinitychromatography.

To address direct binding between the two proteins, agarose-beadconjugated GST (negative control) or GST-target protein was incubatedwith cell lysate or recombinant protein in radioimmunoprecipitationassay (RIPA) buffer (NaCl, 25 mM Tris-Cl, 1% NP-40, 1% sodiumdeoxycholate, 0.1% SDS, protease inhibitor mixture) for 1 hour at atemperature of 4° C. for a reaction.

Immunoprecipitation (IP) assay was performed with cell lysate orrecombinant protein with RIPA buffer. The whole lysates were incubatedwith suitable antibodies for 2 hours at a temperature of 4° C. for areaction, and then, the mixtures were added with A/G-agarosebeads-conjugated secondary antibody (Invitrogen, Carlsbad, Calif., USA)for 2 hours. After incubation for a reaction, the mixtures were washedusing RIPA buffer twice, and precipitated proteins were determined bywester blot analysis.

4. Western Blot Analysis

To carry out western blot analysis, proteins were thermally inactivatedin RIPA buffer (heat treatment for 7 minutes at a temperature of 95°C.), and then, the resulting proteins were applied to SDS-PAGE, followedby western blot analysis according to a known method in the art (J.Clin. Oncol. 16, 1207-1217, 1998; Nat. Rev. Cancer 2, 489-501, 2002).Antibodies used herein, e.g., HA (sc-7392), His (sc-8036), GFP(sc-9996), GST (sc-138), Actin (se-1616), and p19/ARF (sc-32748), werepurchased from Santa Cruz Biochnology, anti-p14/ARF (MAB3782) waspurchased from Millipore, and FLAG-M2 and anti-C-Myc (M5546) werepurchased from Sigma Aldrich. In addition, anti-AIMP2 was provided fromProfessor. Kim, Sung-Hoon (Seoul Nat. Univ.).

5. Screening of DX2-p14/ARF Binding Inhibitor

As shown in FIGS. 2A, 2B, and 3A, SLCB050 was obtained as a compoundthat selectively inhibits DX2-p14/ARF binding. In particular, SLCB050only blocked the interaction of DX2 and AIMP2 as shown in FIG. 2C, butnot on p53-AIMP2 or DX2 binding as shown in FIG. 3B, and p14/ARF asshown in FIG. 3C. In this regard, it was confirmed that SLCB050 would beinteracted with DX2-specific region.

Synthesis of SLCB050 [(7S)-(+)-3-(2-Furanyl)-acrylic acid,8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl-ester]

[Step 1]

Under N₂ gas, trans-3-(2-furanyl)acrylic acid (560 mg, 4.06 mmol, 1 eq),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 1.17g, 6.09 mmol, 1.5 eq), and 4-(dimethylamino)pyridine (4-DMAP, 198 mg,1.62 mmol, 0.4 eq) were added to a 100 mL round-bottom flask, and then,dissolved in anhydrous dichloromethane (100 ml). (S)-(+)-decursinol (1g, 4.06 mmol, 1 eq, KR 0715206) was added to the mixed solution,followed by being stirred at room temperature for 5 hours andconcentrated under reduced pressure. The filtrate was then separated bysilica gel column (ethylacetate: n-hexane=gradient elution to 1:3 from1:8), thereby obtaining (7S)-(+)-3-(2-furanyl)-acrylic acid,8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl-ester(SLCB050) having the following material properties:

yield: 73.6%; orange solid-phase; mp: 96.1° C.; R_(f)=0.62(n-hexane:ethyl acetate=1:1); [α]_(D) ²⁵+62.4 (c=3, CHCl₃);

¹H NMR (400 MHz, CDCl₃): δ_(H) 7.64 (1H, s, H-6′), 7.58 (1H, d, J=9.6Hz, H-4), 7.56 (1H, d, J=16.0 Hz, H-3′), 7.41 (1H, d, J=1.6 Hz, H-7′),7.17 (1H, s, H-5), 6.82 (1H, s, H-10), 6.55 (1H, d, J=1.6 Hz, H-8′),6.23 (1H, d, J=9.6 Hz, H-3), 6.13 (1H, d, J=16.0 Hz, H-2′), 5.17 (1H, t,J=4.4 Hz, H-7), 3.23 (1H, dd, J=4.4, 17.6 Hz, H-6a), 2.92 (1H, dd,J=4.4, 17.6 Hz, H-6b), 1.42 (3H, s, CH₃-8), 1.38 (3H, s, CH₃-8);

¹³C NMR (100 MHz, CDCl₃) δ_(C) 166.3 (C-1′), 161.3 (C-2), 156.3 (C-9a),154.1 (C-10a), 144.8 (C-4′), 144.5 (C-6′), 143.1 (C-4), 135.8 (C-3′),128.7 (C-5), 117.0 (C-2′), 115.6 (C-5a), 113.3 (C-3), 112.9 (C-7′),112.9 (C-4a), 107.2 (C-8′), 104.7 (C-10), 76.6 (C-8), 70.0 (C-7), 27.8(C-6), 24.8 (CH3-8), 23.3 (CH3-8); and

ESI-MS: m/z=389 [M+Na]⁺.

6. Synthesis of HJH141204 and HJH141206

As analogous compounds to the previously screened SLCB050, HJH141204,HJH141206, and SLCB36 were each synthesized.

As shown in FIGS. 3D and 3E, SLCB050, HJH141204, and HJH141206 showedinhibitory effects on DX2-p14/ARF binding and did not affect theinteraction of p53-p14/ARF, whereas SLCB36 did not show inhibitoryeffect on both binding. Accordingly, it was confirmed that inclusion ofa ribose ring structure in the compounds is required for the bindinginhibition.

1) Synthesis of HJH141206: (7S)-(+)-2-(Furan-2-yl)vinylcarbamic acid,8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl-ester

[Step 1] Trans-3-(2-furanyl)acrylic acid (500 mg, 3.37 mmol) wasdissolved in 20 mL of dry benzene in a round-bottom flask, and then,triethyamine (TEA, 234 μl, 1.683 mmol) and diphenyl phosphoryl azide(DPPA, 362 μl, 1.683 mmol) were add thereto for a reaction at atemperature of 80° C. for 3 hours. After an extraction process usingwater and ethylacetate was performed on the mixed solution, adehydration process using sodium sulfate was performed thereon, followedby being concentrated under reduced pressure.

[Step 2] The resulting product was dissolved again in dry benzene, andthen, was heated to reflux at a temperature of 80° C. for one day.

[Step 3] (S)-(+)-decursinol (207 mg, 0.841 mmol) was added thereto, andTEA (281 μl, 2.019 mmol) and 4-(dimethylamino)pyridine (DMAP, 82 mg,0.673 mmol) were also added thereto for a reaction at a temperature of80° C. for 3 hours. The reaction solution was concentrated under reducedpressure, and the filtrate was then separated by silica gel column(ethylacetate: n-hexane=gradient elution to 1:3 from 1:8), therebyobtaining (7S)-(+)-2-(Furan-2-yl)vinylcarbamic acid,8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl-ester(HJH141206) having the following material properties:

yield: 69.7%; brown solid-phase; mp: 58.5° C.; R_(f)=0.46(n-hexane:ethyl acetate=1:1); [α]_(D) ²⁵+79.6933 (c=3, CHCl₃);

¹H NMR (300 MHz, DMSO-d₆): δ_(H) 9.894 (d, J=10.2 Hz, 1H), 7.920 (d,J=9.6 Hz, 1H), 7.493 (s, 2H), 6.984 (dd, J=10.2, 14.7 Hz, 1H), 6.798 (s,1H), 6.401-6.383 (m, 1H), 6.264 (d, J=9.6 Hz, 1H), 6.190 (d, J=3.3, 1H),5.912 (d, J=14.4 Hz, 1H), 5.056 (t, J=3.6 Hz, 1H), 3.254 (dd, J=4.2,18.0 Hz, 1H), 2.917 (dd, J=3.3, 17.7 Hz, 1H), 1.384 (s, 3H, CH₃), 1.313(s, 3H, CH₃);

¹³C NMR (100 MHz, DMSO-d₆) δ_(C) 160.3, 155.8, 153.5, 153.1, 151.5,144.1, 141.1, 129.6, 123.9, 115.7, 112.7, 112.6, 111.5, 105.2, 103.5,99.9, 76.8, 70.2, 27.3, 24.3, 23.7; and

ESI-MS: m/z=382 [M+H]⁺.

2) Synthesis of HJH141204: (7R)-(−)-2-(Furan-2-yl)vinylcarbamic acid,8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl-ester

[Step 1] As shown in the reaction formula above, (+)-decursinol (1, 85g, 0.35 mol) and triphenylphosphine (226 g, 0.87 mol) were added to around-bottom flask, and acetonitrile (600 mL) and carbon tetrachloride(600 mL) were dissolved therein at a ratio of 1:1. Then, the mixedsolution was refluxed at temperatures of 50-60° C. for 2 hours. Abouthalf of the filtrate was concentrated under reduced pressure, followedby being separated by silica gel column, thereby obtaining8,8-domethyl-8H-pyrano[3,2-g]chromen-2-one(8) as a sample in theexperiment having the following material properties:

yield: 98.6%; white solid-phase; m.p: 124° C.; R_(f)=0.62(n-hexane:ethyl acetate=1:1);

¹H NMR (400 MHz, CDCl₃): δ_(H) 7.583 (d, J=9.52 Hz, 1H), 7.049 (s, 1H),6.711 (s, 1H), 6.340 (d, J=10.0 Hz, 1H), 6.213 (d, J=9.52 Hz, 1H), 5.691(d, J=9.76 Hz, 1H), 1.467 (s, 6H, CH₃X2); and

ESI-MS: m/z=229 [M+H]⁺.

[Step 2] 15% sodium hypochlorite (60 mL) and 0.05 M sodium phosphatedibasic (24 mL) were added to a round-bottom flask, and the reactionsolution was adjusted using 1N sodium hydroxide solution or 1Nhydrochloride solution to have pH of 11.3. Then, to the reactionsolution, a solution in which 8,8-dimethyl-8H-pyrano[3,2-g]chromen-2-on(8, 1.3 g, 5.7 mmol) and (R,R)-(−),N,N′-bis(3,5-di-tertbutylsalicylidene)-1,2,-cyclohexanediamino manganese (III) chloride(Jacobsen catalyst, 69.9 mg, 0.11 mmol) were dissolved indichloromethane (15 ml) was added. The mixed reaction solution wasstirred at a temperature of 0° C. for about 7 hours. An extractionprocess was performed on the mixed reaction solution by usingdichloromethane, and an organic layer obtained therefrom was washed withwater, wherein the organic layer was reddish brown. After the organiclayer was dried over anhydrous magnesium sulfate, the filtrate wasconcentrated under reduced pressure. To obtain pure products, theconcentrated solution was separated by silica gel column, therebyobtaining (6R,7R)-6,7-epoxy-8,8-dimethyl-6H-pyrano[3,2-g]chromen-2-on(9)having the following material properties:

yield: 56.3%; white solid-phase; m.p: 145.2° C.; R_(f)=0.32(n-hexane:ethyl acetate=1:1); [α]_(D) ²⁵+201.8 (c=3, CHCl₃);

¹H NMR (400 MHz, CDCl₃): δ_(H) 7.643 (d, J=9.52 Hz, 1H), 7.470 (s, 1H),6.754 (s, 1H), 1H), 6.265 (d, J=9.52 Hz, 1H), 3.976 (d, J=3.88 Hz, 1H),3.551 (d, J=3.88 Hz, 1H), 1.609 (s, 3H, CH₃), 1.312 (s, 3H, CH₃); and

ESI-MS: m/z=245 [M+H]⁺.

[Step 3] As shown in the reaction formula above,(6R,7S)-6,7-epoxy-8,8-dimethyl-6H-pyrano[3,2-g]cyromen-2-on (9, 600 mg,2.456 mmol) was dissolved in tetrahydrofuran in a round-bottom flask.Then, sodium cyanoborohydride and borane trifluoride diethyl etheratewas added thereto. The mixed reaction solution was stirred at atemperature of 0° C. for 30 minutes. The filtrate of the mixed reactionsolution was concentrated under reduced pressure, followed by beingseparated by silica gel column, thereby obtaining(−)-decursinol[decursinol;(7R)-7-hydroxy-8,8-dimethyl-8H-pyrano[3,2-g]chromen-2-on (10)] as asample in the experiment having the following material properties:

yield: 98.6%; white solid-phase; m.p: 135.6° C.; R_(f)=0.179(n-hexane:ethyl acetate=1:1); [α]_(D) ²⁵−18.4 (c=4, CHCl₃);

¹H NMR (400 MHz, CDCl₃): δ_(H) 7.579 (d, J=9.5 Hz, 1H), 7.180 (s, 1H),6.780 (s, 1H), 6.219 (d, J=9.52 Hz, 1H), 3.876 (d, J=5.1 Hz, 1H), 3.112(dd, J=4.8 Hz, 16.7 Hz, 1H), 2.837 (dd, J=5.6 Hz, 16.6 Hz, 1H), 1.397(s, 3H, CH₃), 1.367 (s, 3H, CH₃); and

ESI-MS: m/z=247 [M+H]⁺.

[Step 4] Trans-3-(2-furanyl)acrylic acid (500 mg, 3.37 mmol) wasdissolved in 20 mL of dry benzene in a round-bottom flask, and then, TEA(234 μl, 1.683 mmol) and DPPA (362 μl, 1.683 mmol) were add thereto fora reaction at a temperature of 80° C. for 3 hours. After an extractionprocess using water and ethylacetate was performed on the mixedsolution, a dehydration process using sodium sulfate was performedthereon, followed by being concentrated under reduced pressure.

[Step 5] The resulting product was dissolved again in dry benzene, andthen, was heated to reflux at a temperature of 80° C. for one day.

[Step 6] (−)-decursinol (207 mg, 0.841 mmol) synthesized in step 3 wasadded thereto, and TEA (281 μl, 2.019 mmol) and4-(dimethylamino)pyridine (DMAP, 82 mg, 0.673 mmol) was added theretofor a reaction at a temperature of 80° C. for 3 hours. The reactionsolution was concentrated under reduced pressure, and the filtrate wasthen separated by silica gel column (ethyl acetate: n-hexane=gradientelution to 1:3 from 1:8), thereby obtaining(7R)-(−)-2-(Furan-2-yl)vinylcarbamic acid,8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl-ester(HJH141204) as a sample in the experiment having the following materialproperties:

yield: 80.9%; brown solid-phase; mp: 58.1° C.; R_(f)=0.46(n-hexane:ethyl acetate=1:1); [α]_(D) ²⁵−79.6467 (c=3, CHCl₃);

¹H NMR (300 MHz, DMSO-d₆): 9.896 (d, J=10.2 Hz, 1H), 7.902 (d, J=9.6 Hz,1H), 7.477 (s, 2H), 6.992 (dd, J=10.2, 14.4 Hz, 1H), 6.790 (s, 1H),6.396-6.387 (m, 1H), 6.255 (d, J=9.6 Hz, 1H), 6.185 (d, J=3.3 Hz, 1H),5.919 (d, J=14.4 Hz, 1H), 5.059 (t, J=3.7 Hz, 1H), 3.251 (dd, J=4.2,18.0 Hz, 1H), 2.922 (dd, J=3.3, 18.0 Hz, 1H), 1.384 (s, 3H, CH₃), 1.313(s, 3H, CH₃);

¹³C NMR (100 MHz, DMSO-d₆) δ_(C) 160.3, 155.9, 153.6, 153.2, 151.6,144.0, 141.1, 129.6, 124.0, 115.7, 112.7, 112.6, 111.5, 105.2, 103.5,100.0, 76.9, 70.3, 27.3, 24.3, 23.7; and

ESI-MS: m/z=382 [M+H]⁺.

3) SLCB36 Compound

EXAMPLE 2 Evaluation of Anticancer Effect

1. Cell Culture

A549, HCT116, H1299, and HEK293 cell lines were each purchased fromAmerican Type culture collection (ATCC, Manassas, Va.), and cultured inRPMI-1640 medium or Dulbecco modified Eagle medium (DMEM) supplementedwith 10% fetal bovine serum (FBS) and 1% antibiotics. NSCLC cell lines(NCI-H23, NCI-H322, NCI-H358, and NCI-H460) purchased from ATCC and SCLCcell lines (NCI-H69, NCI-H128m and NCI-H146) purchased from Korean Cellline Bank (KCLB, Seoul, Korea) were cultured in RPMI-1640 mediumsupplemented with 10% FBS. Mouse embryonic fibroblast (MEF) cells wereisolated from 14.5 day embryos using a standard protocol and cultured inDMEM medium supplemented with 15% FBS and 1% antibiotics.

2. Preparation of Mice

All experimental procedures using laboratory animals were approved bythe animal care committee of Pusan National University. DX2 (C57/BL6)and K-RasLA2 (C57/BL6) mice were obtained from Dr. Kim, Sung-Hoon andProfessor. Choi, Kang-Ryeol (Yonsei University), respectively, anddouble Tg mice were obtained by cross-breeding of DX2 and K-RasLA2 mice.Before experiment, all mice were maintained under temperature- andlight-controlled conditions (20-23° C., 12 h/12 h light/dark cycle), andprovided autoclaved food and water.

3. Recombinant Proteins, Immunoprecipitation and GST Pull-Down Assays

In the same manner as in Example 1, recombinant proteins, IP assay, GSTpull-down assay, and western blot analysis were performed.

4. MTT Assay

By MTT assay, anticancer effects were confirmed based on viability oftumor cells. That is, cells were incubated in 0.5 mg/ml of MTT solutionfor 4 hours at a temperature of 37° C. for a reaction, and then,formazen products formed therefrom were dissolved in dimethyl sulfoxide(DMSO). Then, absorbance thereof was measured at 540 nm by using aspectrometer.

5. Drug Treatment In Vivo

DK (5 month-old, N=6) mice were administered with carrier, SLCB050 (5 or10 mg/kg), Adriamycin (1, 2.5, or 5 mg/kg), and a combination of SLCB050and Adriamycin by intraperitoneal (i.p.) injection. After termination ofthe experiment of each group, mice were dissected and isolated lungtissues. For xenograft, 1×10⁷ H446 cells (ATCC) were seeded in nude ice.After 4 weeks, tumor bearing mice were injected with Adriamycin,SLCB0505, or a combination thereof for 6 weeks. Every weeks, tumorvolume and body weight were measured.

6. Histological Analysis

After dissection of mice, tissues were fixed using 10% formalin (in PBS)for 24 hours, and embedded in paraffin blocks according to a basictissue processing procedure. For histological analysis, embedded tissueswere cut for 5 μM by Leica microtome (Wetzlar, Germany) and transferredonto adhesive-coated slides (Marienfeld laboratory glassware, Germany).After deparaffin and rehydration, sections were then stained with H&Efor routine examination.

For IHC staining, rehydrated tissue sections were incubated withantibodies to Ki-67 (Abcam, ab15580), pan-keratin (Sigma, C2931),pro-surfactant C (Millipore, AB3786), NSE (DAKO, IS612), and HER2/Neu(DAKO, A0458) for a reaction. Antigen retrieval was performed using 10mM sodium citrate (pH 6.0) twice at a temperature of 95° C. for 10minutes each, and endogenous peroxidase activity was blocked with 3%hydrogen peroxidase for 10 min. Then, the slides were dehydratedfollowing a standard procedure and sealed with cover glass usingmounting solution. TUNEL reaction was done as described in the manualfor In Situ Cell Death Detection Kit, POD (Hoffmann-La Roche Ltd, Basel,Swizerland).

7. Analysis of Tumor Incidence and Area

To evaluate tumor incidence, lung tissues of each mouse was fixed andembedded in paraffin. 5 sections from each mouse were examined by 3independent investigator, who counted tumor. In addition, tumor area wascalculated by tumor occupied area in total lung area using photoshopsoftware.

8. Experiment Results

As shown in FIGS. 4A and 4B, SLCB050 treatment blocked the interactionof DX2-p14/ARF, and DX2 and p14/ARF were localized in nucleus. Inaddition, the increase of p14/ARF in SLCB050-treated H1299 and H69 celllines was observed as shown in FIG. 4C while reduction of DX2 inp14/ARF-deficient cell lines (H322, H460, and A549) was observed asshown in FIG. 4D. In addition, p14/ARF-deficient cell lines wereresistant to SLCB050 as shown in FIG. 5, whereas SCLC cell lines weresensitive to SLCB050. In particular, SLCB050 completely suppressed theH128 cell growth as shown in FIG. 6. In addition, HJH141204 andHJH141206 also suppressed cell viability as shown in FIG. 7, andaccordingly, it was confirmed that rumor cell growth suppression wasachieved by compound-DX2 binding.

It was evaluated whether SLCB050 was able to restore the sensitivity tothe anticancer drug. As shown in FIG. 8A, resistance to GN 25(synthesized as described in KR 1298168) in DX2 and DK MEF was abolishedby co-treatment of SLCB050. In addition, as shown in FIG. 8B, resistanceto Adriamycin in DX2 and DK MEF was abolished by co-treatment ofAdriamycin (Adr) and SLCB050. In addition, as shown in FIG. 8C, SCLCcell line H69 was partially responded to SLCB050. However, as shown inFIG. 8C, p14/ARF deficient H322 did not show synergic response tocombination treatment with SLCB050.

As shown in FIG. 9A, the tumor xenograft model using H446 was resistantto Adriamycin and partially responded to SLCB050. In addition, as shownin FIG. 9B, the increased tumor volume in the tumor xenograft modelusing H446 was moderately suppressed by SLCB050 injection (10 mg/kg,three times/week). In addition, the tumor inhibition effect was observedby injection of 5 mg/kg of Adriamycin (three times/week), but it evokedrapid weight loss and death. In contrast, combinational treatment withSLCB050 (10 mg/kg) showed more obvious anticancer effect as shown inFIG. 9B, despite low dosage (2.5 mg/kg) of Adriamycin treatment. Indeed,combinational treatment of Adriamycin and SLCB050 could obviously inducep53 expression in primary tumor cells obtained from DX2 or DK mouse asshown in FIG. 9C. These results indicate that inhibition of DX2 couldenhance anticancer drug sensitivity through re-activation of p14/ARF.

To evaluate the anticancer effect of SLCB050 in a mouse model, SLCB050was injected into DK mice according to an experimental schedule as shownin FIG. 10A. Combinational treatment of SLCB050 (10 mg/kg) and low dose(2.5 mg/kg) Adriamycin significantly suppressed tumor progression asshown in FIGS. 10B and 10C without significant weight loss. However, asshown in FIG. 10B, non-toxic dose of Adriamycin did not show anticancereffect on the mouse model.

More detailed histological analysis showed that SCLC region was moreobviously erased by combinational treatment of Adriamycin and SLCB050 asshown in FIG. 11. As shown in FIG. 12A, under DX2-reduced condition uponSLCB050 treatment, apoptotic tumor cells were obviously increased asshown in FIGS. 12B and 12C. These results indicate that DX2 produced byaberrant splicing of AIMP2 promotes tumor progression, in particular,small cell lung cancer, via direct interaction and inhibition of p14/ARFas shown in FIG. 13. Thus, it was confirmed that small cell lung cancerwould be treated by the compound inhibiting DX2-p14/ARF binding.

EXAMPLE 3 Comparative Measurement of AIMP2-DX2 Expression Levels in SCLCand NSCLC Cells

1. Cell Culture

NSCLC cell lines (A549,H1299, NCI-H23, NCI-H322, NCI-H358, andNCI-H460), HCT116 cell lines, and HEK293 cell lines were available fromAmerican Type Culture Collection (Manassas, Va.), and cultured inRPMI-1640 medium or DMEM supplemented with 10% FBS and 1% antibiotics.

NCI-H69, NCI-H128, and NCI-H146, which are SCLC cell lines, werepurchased from Korean Cell Line Bank (Seoul, Korea), and cultured in aRPMI-1640 medium supplemented with 10% FBS.

2. Measurement of AIMP2-DX2 Expression Levels

Cells cultured in RIPA buffer (150 mM NaCl, 25 mM Tris-Cl, 1%NonidetP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate,protease inhibitor cocktail) were suspended to separate proteinstherefrom.

A sample including the separated proteins was thermally inactivated at atemperature of 95° C. for 7 minutes, and then, the resulting proteinswere applied to SDS-PAGE, followed by western blot analysis according toa known method in the art (T. Mahmood, P. C. Yang. N. Am. J. Med.Science. 4: 429-434, 2012).

Commercially available Actin antibodies (sc-1616, Santa CruzBiotechnology) were used, and AIMP2 and AIMP2-DX2 antibodies wereprepared according to a conventional antibody preparation method.

As shown in FIG. 14, AIMP2-DX2 was similarly expressed in NSCLC cellsand SCLC cells without a significant difference.

3. Measurement of AIMP2-DX2 Transcription

To measure ATMP2-DX2 transcription levels, NSCLC cells, such as A549,H1299, H23, H322, and H460, and SCLC cells, such as H146 and H69, wereused. Primer sequences of SEQ ID NO: 1 or 2 listed in Table 1 were usedto amplify ATMP2-DX2, and primer sequences of SEQ ID NO: 3 or 4 listedin Table 1 were used to amplify GAPDH.

Consequently, as shown in FIG. 15, AIMP2-DX2 transcription levels werenot different from those in SCLS cells and NSCLC cells.

TABLE 1 SEQ ID Name of primer Sequence NO Forward ATMP2-DX25′- AACGTGCACGGCAGGAGCTAC -3′ 1 Reverse ATMP2-DX25′- CCAGCTGATAGTCTTGGCGGG -3′ 2 Forward GAPDH5′- ATCTTCCAGGAGCGAGATCCC -3′ 3 Reverse GAPDH5′- AGTGAGCTTCCCGTTCAGCTC -3′ 4

EXAMPLE 4 Measurement of AIMP2-DX2 Autoantibody Levels Serum of PatientsWith SCLC and NSCLS

Sera of health individuals and patients with SCLC and NSCLC wereavailable from Bucheon Hospital (schbc-biobank-2011-003). To measureanti-AIMP2-DX2 antibody levels, autoantibody levels were measuredaccording to the experiment protocol shown in FIG. 16. RecombinantAIMP2-DX2, Lamin A, and Snail were each attached onto a nitrocellulosemembrane (0.5 ng/well). Here, each membrane was incubated with a serumsample, which was diluted with blocking buffer at a ratio of 1:1000, fora reaction for 1 hour, and sequentially with (HRP)-conjugated anti-humanantibodies (1:20,000) for 30 minutes. Then, sites on which humanantibodies were attached were visualized by ECL in terms ofchemiluminescence and X-ray film exposure.

As a result, as shown in FIG. 17, AIMP2-DX2 autoantibodies were notdetected at all in control group (healthy individuals) while they weredetected in a group of patients with NSCLC or SCLC. In particular, inthe case of a group of patients with SCLC (8 out of 10 cases) AIMP2-DX2autoantibodies were detected, and thus, it was confirmed that AIMP2-DX2autoantibodies were significantly highly detected in serum of a group ofpatients with SCLC.

However, SLCB050 of the present invention can be formulated in variousforms according to purposes. In the following, embodiments offormulations using the compound of the present invention as an effectivecomponent are provided, but the present invention is not limitedthereto.

FORMULATION EXAMPLE 1 Tablet (Direct Pressurization)

5.0 mg of SLCB050 was filtered through a sieve, and then, mixed with14.1 mg of lactose, 0.8 mg of Crospovidone USNF, and 0.1 mg of magnesiumstearate. A pressure was applied thereto to prepare the mixture in formof a tablet.

FORMULATION EXAMPLE 2 Tablet (Wet Granulation)

5.0 mg of SLCB050 was filtered through a sieve, and then, mixed with16.0 mg of lactose and 4.0 mg of starch. A solution in which 0.3 mg ofPolysorbate 80 was dissolved in pure water was added to the mixture inan appropriate amount, and then, the resulting solution was subjected tograin refining. After being dried, grains were filtered through a sieve,and then, mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg ofmagnesium stearate. A pressure was applied thereto to prepare the grainsin form of a tablet.

FORMULATION EXAMPLE 3 Powder and Capsule

5.0 mg of SLCB050 was filtered through a sieve, and then, mixed with14.8 mg of lactose, 10.0 mg of polyvinyl pyrrolidone, and 0.2 mg ofmagnesium stearate. The mixture was added to hard Gelatin Capsule Size 5by using an appropriate device, thereby preparing the mixture in form ofa capsule.

FORMULATION EXAMPLE 4 Injection

100 mg of SLCB050, 180 mg of mannitol, 26 mg of Na₂HPO₄12H₂O, and 2.974mg of distilled water were mixed together. A transparent glass ampoulewas filled with the mixed solution, and then, sealed under an upperlattice by dissolving the glass. A sterilization process was performedthereon through autoclave at a temperature of 120° C. for at least 15minutes, thereby preparing the mixture in form of injection.

It should be understood that embodiments described herein should beconsidered in a descriptive sense only and not for purposes oflimitation. Descriptions of features or aspects within each embodimentshould typically be considered as available for other similar featuresor aspects in other embodiments.

[Sequence List Free Text]

SEQ ID NO: 1 or 2 is a base sequence of a pair of primers for amplifyingAIMP2-DX2.

SEQ ID NO: 3 or 4 is a base sequence of a pair of primers for amplifyingGAPDH.

SEQ ID NO: 5 is an amino acid sequence of an AIMP2-DX2 protein.

SEQ ID NO: 6 is an amino acid sequence of an AIMP2-DX2 protein.

What is claimed is:
 1. A method of treating a cancer disease in asubject, comprising: providing a pharmaceutical composition comprising,as an active ingredient, a compound represented by Formula 3:

and administering the pharmaceutical composition to the subject, whereinthe cancer disease is treated, wherein the cancer disease is selectedfrom the group consisting of lung cancer, colorectal cancer, livercancer, stomach cancer, esophageal cancer, pancreatic cancer,gallbladder cancer, kidney cancer, bladder cancer, prostate cancer,testis cancer, uterine cervical cancer, endometrial cancer,choriocarcinoma, ovarian cancer, breast cancer, thyroid cancer, braincancer, head and neck cancer, malignant melanoma, lymphoma, andhematologic malignancy.
 2. The method of claim 1, wherein the lungcancer is non-small cell lung cancer or small cell lung cancer.
 3. Amethod of inhibiting drug resistance of an anticancer drug in a subject,comprising: providing a pharmaceutical composition comprising, as anactive ingredient, a compound represented by Formula 3:

and administering the pharmaceutical composition to the subject, whereindrug resistance of the anticancer drug is inhibited.
 4. The method ofclaim 3, wherein the anticancer drug is selected from the groupconsisting of Adriamycin, Capecitabine, Caboplatin, Cisplatin,Oxaliplain, Cyclophosphamide, Docetaxel, Paclitaxel, Doxorubicin,Daunorubicin, Epirubicin, Idarubicin, Valrubicin, Mitoxantrone,Curcumin, Gefitinib, Erlotinib, Irinotecan, Topotecan, Vinblastine,Vincristine, Gemsitabin, Methotrexate, Trastzumab, Vinorelbine,Fluorouracil, and 3-(5,8-dimethoxy-1,4-dioxonaphthalene-2-ylthio)propanoic acid.
 5. The methodof claim 3, wherein the compound inhibits binding between DX2 andp14/ARF proteins so that drug resistance of the anticancer agent isinhibited and anticancer effect of the anticancer agent is enhanced.